CN115998815B - Traditional Chinese medicine composition for treating acute coronary syndrome complicated with renal insufficiency and application thereof - Google Patents
Traditional Chinese medicine composition for treating acute coronary syndrome complicated with renal insufficiency and application thereof Download PDFInfo
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a traditional Chinese medicine composition for delaying atherosclerosis and renal insufficiency progression and application thereof. The traditional Chinese medicine composition comprises the following raw material medicines: cistanche, medlar, red paeony root, allium macrostemon, ginseng and ligusticum wallichii. The invention also discloses a preparation method of the traditional Chinese medicine composition and application of the traditional Chinese medicine composition in preparation of medicines for delaying atherosclerosis and renal insufficiency progression. The Chinese medicinal composition inhibits apoptosis of vascular endothelial cells and tubular epithelial cells by regulating and controlling NEAT1/miR-139-5p/Fos axes so as to delay atherosclerosis and renal insufficiency progression.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicines. More particularly relates to a traditional Chinese medicine composition for delaying atherosclerosis and renal insufficiency progression and application thereof.
Background
Heart disease and kidney disease are serious diseases in China, have high morbidity and high mortality, and seriously threaten human health. In recent years, modern medical research gradually discovers that heart diseases and kidney diseases have relevance, a plurality of common indexes of chronic kidney diseases are independent risk factors for occurrence and development of heart diseases, clinically both diseases often occur, if the heart diseases and the kidney diseases cannot be effectively controlled in the early stage, the heart diseases and the kidney diseases are extremely easily developed into heart-kidney syndrome (CRS), functional damages of one organ to the other organ cannot be compensated, the heart diseases and the kidney diseases are mutually causal, malignant circulation is formed, and finally the heart and kidney functions are commonly damaged and exhausted, wherein vascular endothelial cell apoptosis and renal tubular epithelial cell apoptosis are important pathological processes, and how to delay the occurrence of the pathological processes through a combination means of traditional Chinese medicine and western medicine, so that the development of atherosclerosis and renal insufficiency is a key problem to be solved urgently.
Disclosure of Invention
Aiming at the technical problems, an object of the invention is to provide a novel traditional Chinese medicine composition which can effectively delay the progression of atherosclerosis and renal insufficiency.
The invention also aims at providing a preparation method of the traditional Chinese medicine composition.
The invention also aims to provide the application of the traditional Chinese medicine composition in preparing medicines for delaying the progression of atherosclerosis and renal insufficiency.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a traditional Chinese medicine composition for effectively delaying the progression of atherosclerosis and renal insufficiency, which comprises the following raw materials: cistanche, medlar, red paeony root, allium macrostemon, ginseng and ligusticum wallichii.
Preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 6-40 parts of cistanche, 10-40 parts of medlar, 3-20 parts of red paeony root, 6-20 parts of allium macrostemon, 3-15 parts of ginseng and 6-30 parts of ligusticum wallichii.
The traditional Chinese medicine composition is mainly used for patients with atherosclerosis and renal insufficiency, and has symptoms of chest pain and chest distress, or pain induced by shoulder and back, purple and dark mouth and lips, soreness and weakness of waist and knees, frequent nocturia and the like. The inventor discovers that the traditional Chinese medicine composition can obviously improve the clinical curative effects of atherosclerosis and renal insufficiency patients through massive screening in the early stage of heart disease and kidney disease, wherein the pathological changes of vascular endothelial cell apoptosis and renal tubular epithelial cell apoptosis exist, and the mechanism is that the H is lightened by regulating the lncRNA NEAT1/miR-139-5p/Fos axis 2 O 2 Damage to HUVEC and HK-2 cells, inhibit ROS formation, reduce expression of AP-1, caspase 3 and Caspase9, and inhibit apoptosis of HUVEC and HK-2 cells, thereby playing roles in delaying atherosclerosis progression and protecting kidney.
In the prescription of the invention, the monarch drug is cistanche deserticola. Cistanche deserticola: sweet, salty and warm in taste, enters kidney and large intestine meridian, tonifies kidney and benefits essence, moistens intestine and relieves constipation, and the "materia medica Hui Yan" is: cistanche salsa, a medicine for nourishing life gate, replenishing kidney qi and replenishing essence and blood. Yang tonifying, dryness preventing, wen Tongshen yang tonifying, kidney deficiency preventing; tonify yin but not greasy, moisten intestines and dredge abdomen to treat constipation.
The ministerial drugs comprise fructus Lycii, radix Paeoniae Rubra, and Bulbus Allii Macrostemi. Wolfberry fruit: sweet in taste, calm, good in liver and kidney meridians, nourishing liver and kidney, replenishing vital essence and improving eyesight. Radix paeoniae rubra: bitter and slightly cold. It has the functions of invigorating liver and spleen, clearing away heat and cooling blood, promoting blood circulation to disperse blood clots. Allium macrostemon: pungent and bitter in flavor, warm in nature, enters lung, stomach and large intestine meridians, and is pungent in flavor, warm in nature, unblocking yang and resolving hard mass.
The adjuvant drug is ginseng and rhizoma ligustici wallichii. Ginseng: sweet and slightly bitter taste, slightly warm nature, and spleen, lung, heart and kidney meridians entered, and is mainly used for treating primary qi and desire to be removed, listlessness and pulse faint caused by serious illness, chronic illness, blood loss and liquid removal, and is suitable for insomnia and dreaminess due to heart qi deficiency, palpitation and amnesia, and asthenia and hyperhidrosis. The medicines are mutually compatible, and the medicine has the effects of activating and tonifying, dispersing blood stasis, warming kidney yang, strengthening tendons and bones, strengthening middle-jiao and qi, resolving phlegm and dampness, and is a preparation for treating both symptoms and root causes of chest stuffiness and heart pain due to kidney deficiency and blood stasis. Ligusticum wallichii: pungent and warm taste, enter liver, gallbladder and pericardium meridian, and are qi-flowing in blood, promoting blood circulation, activating qi-flowing, dispelling wind, relieving pain, being pungent, warm, fragrant and dry, and not only can promote dispersion, but also can promote upward movement to peak top, and blood enters downward movement to blood sea, and has the functions of pungent dispersion, resolving depression, promoting passage, relieving pain, etc.
In the invention, the traditional Chinese medicine composition for delaying the progression of atherosclerosis and renal insufficiency comprises the following raw materials in parts by weight: 30 parts of cistanche, 40 parts of medlar, 15 parts of red paeony root, 10 parts of allium macrostemon, 10 parts of ginseng and 10 parts of ligusticum wallichii.
The traditional Chinese medicine composition for delaying the progression of atherosclerosis and renal insufficiency comprises the following raw materials in parts by weight: 40 parts of cistanche, 30 parts of medlar, 20 parts of red paeony root, 20 parts of allium macrostemon, 15 parts of ginseng and 30 parts of ligusticum wallichii.
The traditional Chinese medicine composition for delaying the progression of atherosclerosis and renal insufficiency comprises the following raw materials in parts by weight: 6 parts of cistanche, 10 parts of medlar, 3 parts of red paeony root, 6 parts of allium macrostemon, 3 parts of ginseng and 6 parts of szechuan lovage rhizome.
The traditional Chinese medicine composition can be adjusted based on the traditional Chinese medicine composition according to clinical symptoms when the traditional Chinese medicine composition is specifically implemented, so that the purposes of delaying atherosclerosis and renal insufficiency progression and improving certain special clinical symptoms are achieved, the adaptability of the traditional Chinese medicine composition is further enhanced, and the treatment effect is improved. These adjustments made on the basis of the basic recipe of the present invention are also within the scope of the present application.
In a second aspect, the present invention provides a method for preparing the above-mentioned Chinese medicinal composition for delaying the progression of atherosclerosis and renal insufficiency, which comprises the following steps:
pulverizing the raw materials for preparing the traditional Chinese medicine composition, mixing, adding 6-10 times of water for soaking for 0.5-2 hours, decocting for 2-3 times, each time for 40-60 minutes, mixing decoctions, filtering, and concentrating the filtrate under reduced pressure until each milliliter of liquid medicine is equivalent to 0.2-3g of crude drug, thus obtaining the traditional Chinese medicine composition; or,
crushing Ginseng radix to obtain Ginseng radix, reflux-extracting with 6-10 times of 60-80% ethanol water solution for 2-3 times (each for 1-2 hr), filtering, collecting the combined filtrates, recovering ethanol, and concentrating the filtrate under reduced pressure to obtain Ginseng radix ethanol extract containing 0.1-1g crude drug per ml of medicinal liquid; mixing the residue with other materials, decocting with 6-10 times of water for 2-3 times for 40-60 min, mixing decoctions, filtering, mixing filtrate with the Ginseng radix ethanol extract, and concentrating into medicinal liquid of 0.2-3 g/ml; or,
pulverizing Ginseng radix into fine powder; mixing the other raw materials, decocting with 6-10 times of water for 2-3 times, each time for 40-60 min, mixing decoctions, filtering, concentrating the filtrate under reduced pressure until the concentration of each milliliter of liquid medicine is equivalent to 0.2-3g of crude drug, and adding Ginseng radix fine powder into the concentrated solution, and mixing.
In a third aspect, the present invention provides a medicament for delaying the progression of atherosclerosis and renal insufficiency, which comprises the above-mentioned Chinese medicinal composition as an active ingredient.
According to actual needs, the medicine can also comprise pharmaceutically acceptable auxiliary materials. Making into various dosage forms such as decoction, granule, pill, capsule, tablet, powder, and oral liquid according to conventional preparation process.
The invention also provides application of the traditional Chinese medicine composition in preparation of medicines for delaying the progression of atherosclerosis and renal insufficiency.
In addition, unless specified, all the raw materials of the traditional Chinese medicine composition can be obtained commercially, and any range described in the invention comprises any value between the end values and any sub-range formed by any value between the end values or any value between the end values can achieve the aim of delaying the progression of atherosclerosis and renal insufficiency.
The beneficial effects of the invention are as follows:
heart disease and kidney disease are serious diseases in China, have high morbidity and high mortality, and seriously threaten human health. In recent years, modern medical research gradually discovers that heart diseases and kidney diseases have relevance, and common indexes of many chronic kidney diseases are independent risk factors for occurrence and development of heart diseases, and clinically both diseases are often accompanied. The traditional Chinese medicine composition disclosed by the invention is strict in formula, can be used for simultaneously improving the injury and failure of two important organs of heart and kidney from the aspect of scientific problems, and can be used for avoiding the occurrence of heart-kidney syndrome, and has the effects of tonifying kidney, strengthening the root, promoting blood circulation, removing blood stasis and dredging and tonifying both from the aspect of traditional Chinese medicine theory. Through research on the medicinal mechanism, the research on the medicinal mechanism shows that the medicinal mechanism possibly reduces H by regulating the lncRNA NEAT1/miR-139-5p/Fos axis 2 O 2 Damage to HUVEC and HK-2 cells, inhibition of ROS production, reduction of AP-1, caspase 3 and Caspase9 expression, inhibition of HUVEC andapoptosis of HK-2 cells, thereby exerting the effects of delaying the progression of atherosclerosis and protecting the kidneys.
Drawings
FIG. 1 shows a differential lncRNA heatmap;
FIG. 2 shows a volcanic lncRNA plot;
FIG. 3 shows differential miRNA heatmaps;
fig. 4 shows a miRNA volcanic plot;
FIG. 5 shows differential mRNA heatmaps;
FIG. 6 shows an mRNA volcanic plot;
FIG. 7 shows GO enrichment of the first 30 (note: green for biological process; blue for cellular composition; red for molecular function);
FIG. 8 shows KEGG functional enrichment of 382 differential mRNAs;
FIG. 9 shows KEGG functional enrichment of 249 upregulated mRNAs;
FIG. 10 shows KEGG functional enrichment of 133 downregulated mRNAs;
FIG. 11 shows a ceRNA regulatory network (note: blue circles are lncRNA; green triangles are miRNA; red diamonds are mRNA);
FIG. 12 shows a screening procedure for the lncRNA NEAT1/miR-139-5p/Fos regulatory axes;
FIG. 13 shows the effect of a Chinese medicinal composition of the present invention on the lncRNA NEAT1/miR-139-5P/Fos axis (note: P <0.05, P <0.01 compared to before treatment);
fig. 14 shows the effect of the inventive composition on patient plasma Fos protein (note: P <0.05, < P <0.01 compared to prior to treatment);
FIG. 15 shows validation of lncRNA NEAT1 binding to miR-139-5P targeting (note: P <0.05, <0.01 compared to cells not transfected with miR-139-5P);
FIG. 16 shows possible binding sites for lncRNA NEAT1 and miR-139-5 p;
FIG. 17 shows validation of miR-139-5P and Fos targeted binding (note: P <0.05, P <0.01 compared to cells not transfected with miR-139-5P);
FIG. 18 shows the possible binding sites for miR-139-5p and Fos;
FIG. 19 shows the OD values and cell inhibition rates of CCK8 tested against HUVEC and HK-2 cells of each group (note: P <0.05, # P <0.01 compared to control group; #P <0.05, # P <0.01 compared to model group);
FIG. 20 shows flow cytometry to detect cellular ROS levels in groups (note: A: HUVEC control; B: HUVEC model; C: HUVEC treatment; D: HK-2 control; E: HK-2 model; F: HK-2 treatment);
FIG. 21 shows apoptosis rates of HUVEC and HK-2 cells of each group (note: P <0.05, # P <0.01 compared to control group, # P <0.05, # P <0.01 compared to model group);
FIG. 22 shows flow cytometry for detection of apoptosis in groups (note: A: HUVEC control group; B: HUVEC model group; C: HUVEC treatment group; D: HK-2 control group; E: HK-2 model group; F: HK-2 treatment group);
FIG. 23 shows Tunel staining for detection of apoptosis index for each group of HUVEC cells (note: green fluorescence is Tunel-stained apoptotic nuclei and blue fluorescence is DAPI-stained total nuclei);
FIG. 24 shows Tunel staining to detect apoptosis index of HUVEC cells in each group (note: P <0.05, # P <0.01 compared to control group, # P <0.05, # P <0.01 compared to model group);
FIG. 25 shows Tunel staining to detect apoptosis index of each set of HK-2 cells (note: green fluorescence is Tunel-stained apoptotic nuclei and blue fluorescence is DAPI-stained total nuclei);
FIG. 26 shows Tunel staining to detect apoptosis index of HK-2 cells in each group (note: P <0.05, # P <0.01 compared to control group, # P <0.05, # P <0.01 compared to model group);
FIG. 27 shows the effect of the inventive Chinese medicinal composition on the lncRNA NEAT1/miR-139-5P/Fos axes of the groups HUVEC and HK-2 cells (note: P <0.05, # P <0.01 compared to control group;, # P <0.05, # P <0.01 compared to model group);
FIG. 28 shows the effect of a Chinese medicinal composition of the present invention on Caspase 3 and Caspase9 in each of the HUVEC and HK-2 cells (note: P <0.05, # P <0.01 compared to control group; #P <0.05, # P <0.01 compared to model group);
FIG. 29 shows Western Blot detection of HUVEC cellular protein expression levels for each group (note: A: HUVEC control; B: HUVEC model; C: HUVEC treated);
FIG. 30 shows Western Blot detection of protein expression levels of HK-2 cells from each group (note: D: HK-2 control group; E: HK-2 model group; F: HK-2 treatment group).
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.
Example 1A Chinese medicinal composition for delaying the progression of atherosclerosis and renal insufficiency
The formula of the raw materials comprises the following steps: 30g of cistanche, 40g of medlar, 15g of red paeony root, 10g of allium macrostemon, 10g of ginseng and 10g of ligusticum wallichii.
Reflux-extracting radix Ginseng prepared into the Chinese medicinal composition with 8 times of 75% ethanol water solution for 2 times (each for 1 hr), filtering, collecting the combined filtrates, recovering ethanol, and concentrating the filtrate under reduced pressure to obtain radix Ginseng ethanol extract containing 1g crude drug per ml of medicinal liquid; mixing the residue with other materials, decocting with 8 times of water for 2 times and 60 min, mixing decoctions, filtering, mixing filtrate with the Ginseng radix ethanol extract, and concentrating to obtain medicinal liquid equivalent to 3g of crude drug per ml.
Example 2A Chinese medicinal composition for delaying the progression of atherosclerosis and renal insufficiency
The formula of the raw materials comprises the following steps: 40g of cistanche, 30g of medlar, 20g of red paeony root, 20g of allium macrostemon, 15g of ginseng and 30g of szechuan lovage rhizome.
Pulverizing Ginseng radix into fine powder; mixing the other raw materials, decocting with 10 times of water for 3 times, each time for 60 minutes, mixing decoctions, filtering, concentrating the filtrate under reduced pressure to give medicinal liquid equivalent to 3g per ml, and adding Ginseng radix fine powder into the concentrated solution, and mixing.
Example 3A drug for delaying the progression of atherosclerosis and renal insufficiency
The formula of the raw materials comprises the following steps: cistanche salsa 6g, wolfberry fruit 10g, red peony root 3g, allium macrostemon 6g, ginseng 3g and Ligusticum wallichii 6g.
Pulverizing the raw materials for preparing the traditional Chinese medicine composition, mixing, soaking in 6 times of water for 2 hours, decocting for 2 times, each time for 60 minutes, mixing decoctions, filtering, concentrating the filtrate under reduced pressure until the concentration of each milliliter of liquid medicine is equivalent to 3g of crude drug, and obtaining the traditional Chinese medicine composition.
EXAMPLE 4 basic study of the Chinese medicinal composition of the invention to regulate NEAT1/miR-139-5p/Fos axis to inhibit apoptosis of vascular endothelial cells and tubular epithelial cells and delay progression of atherosclerosis and renal insufficiency
1.1 high throughput sequencing and qRT-PCR case collection: 5 cases of patients with acute coronary syndromes and renal insufficiency are selected, the traditional Chinese medicine composition prepared in the embodiment 3 is taken for patients, 1 dose is taken separately in the morning and evening, the treatment course is 4 weeks, peripheral blood mononuclear cells (Peripheral blood mononuclear cells, PBMCs) before and after treatment by using the composition (embodiment 3) are collected for carrying out second-generation high-throughput sequencing, and difference lncRNA, miRNA and mRNA before and after treatment are screened and a ceRNA regulation network is constructed. Meanwhile, the sample size is further enlarged, 20 patients are collected for PBMCs before and after treatment, and the regulation and control effect of the traditional Chinese medicine composition on the lncRNA-miRNA-mRNA axis is verified by utilizing a qRT-PCR technology.
1.2 sample collection: subjects were drawn on an empty stomach before treatment and after 4 weeks of treatment, and 4-8ml of peripheral venous blood was drawn from the patient using EDTA vacuum anticoagulant tube, stored in a refrigerator at 4 ℃ and isolated within 4 hours.
1.3 separation of PBMCs from plasma: extracting plasma after centrifugation of the EDTA blood collection tube; PBMCs were extracted using erythrocyte lysate and TRIzol reagent.
1.4 total RNA extraction and quality control: extracting total RNA with chloroform and isopropanol; 1 μl of the sample was taken, and the concentration and purity of the sample were measured by a NanoDrop2000 spectrophotometer to detect the quality of the RNA sample.
1.5 library construction and high throughput sequencing: usingUltra TM RNA Library Prep Kit for Illumina and Ribo-Zero Magnetic Gold Kit library construction of lncRNA and mRNA sequencing analysis was performed on each sample using a HiSeq 4000 pe150 sequencer. Use->Small RNA Library Prep Setfor Illumina library construction of mirnas. The miRNA of each sample was sequenced using a Hiseq X Ten PE150 sequencer.
1.6 screening of differential genes: the count numbers of lncRNA, miRNA and mRNA in each sample are standardized by using DESeq software, and the differential gene is analyzed by using a Benjamini-Hochberg method and fold difference (FC) is calculated. P value <0.05 and |log2FC| >0.58 were used as criteria for screening for differential genes. Volcanic and thermal maps of differential lncRNA, differential miRNA and differential mRNA were plotted using the "gglot 2" and "phypatmap" packages of R software.
Experimental results:
through transcriptome high-throughput sequencing, research shows that 696 different lncRNAs (314 upregulations, 382 downregulation) exist before and after treatment of the traditional Chinese medicine composition, 86 different miRNAs (74 upregulations, 12 downregulation) and 382 different mRNAs (249 upregulations, 133 downregulation) exist. The thermal and volcanic patterns of the various lncRNA, miRNA and mRNA are shown in fig. 1-6.
1.7 functional enrichment of differential mRNA for GO and KEGG: in order to further define the potential action mechanism of the traditional Chinese medicine composition, the research adopts a DAVID (https:// DAVID. Ncifcrf. Gov /) online database to carry out GO and KEGG functional enrichment on differential mRNA, and the difference is statistically significant when P is less than 0.05.
Experimental results:
GO and KEGG functional enrichment was performed on 382 differential mrnas using the DAVID online database. GO enrichment consists of 3 parts, biological processes (Biological Processes, BP), cellular composition (Cellular Component, CC) and molecular function (Molecular Function, MF). The GO enrichment of the first 30 is shown in fig. 7, and the result shows that BP is mainly involved in biological processes such as platelet activation, blood coagulation, positive regulation of phosphatidylinositol 3-kinase signal transduction, and leukocyte migration; CC mainly involves cell membranes, extracellular space, actin cytoskeleton, etc.; whereas MF is mainly related to the activity of chemokines, guanylate cyclase activity and actin binding etc.
In the KEGG enrichment analysis, 382 different mRNAs were functionally enriched (FIG. 8), and the research shows that the signal path interfered by the traditional Chinese medicine composition mainly comprises aspects of platelet activation, vascular smooth muscle contraction, cytokine receptor interaction, TGF-beta signal path and the like. At the same time KEGG functional enrichment was performed for 249 up-regulated mrnas (fig. 9) and 133 down-regulated mrnas, respectively (fig. 10). In upregulated mRNA, the pathways affected by the inventive compositions include platelet activation, cGMP-PKG signaling pathways, vascular smooth muscle contraction, and TGF-beta signaling pathways. In the case of down-regulation of the KEGG enrichment of mRNA, signaling pathways mainly include Toll-like receptor signaling pathway, TNF signaling pathway, NOD-like receptor signaling pathway, NF-. Kappa.B signaling pathway, MAPK signaling pathway, and the like.
1.8 target Gene prediction and construction of the ceRNA network: and predicting target miRNAs of the differential lncRNAs by using a miRcode database, and taking intersections with the differential miRNAs obtained by sequencing. miRNA targets bound mRNA, and prediction is carried out through TargetScan, miRDB and mirtarBase databases, and only 3 databases have relevant reports, the regulation relationship of miRNA-mRNA is considered to exist. Finally, the predicted mRNA is intersected with the differential mRNA screened by high throughput sequencing. Based on the interactions of lncRNA-miRNA and miRNA-mRNA above, the lncRNA-miRNA-mRNA network was visualized using Cytoscape 3.8.0 software.
Experimental results:
and predicting miRNA targeted lncRNA by using a miRcode database, wherein the miRNA targeted lncRNA and the miRNA have a targeted regulation and control relationship, and the miRNA targeted lncRNA is incorporated into the construction of a ceRNA network. Researches show that 27 lncRNAs in 696 different lncRNAs can find targeted binding miRNAs in a database, and the targeted binding miRNAs with 205 miRNAs is achieved through 2024 lncRNA-miRNA regulation axes. The predicted 205 miRNAs are intersected with 86 miRNAs obtained by high-throughput sequencing screening, so that 3 differential miRNAs (hsa-miR-139-5 p, hsa-miR-126-3p and hsa-miR-135a-5 p) are obtained. Further using TargetScan, miRDB and miRTarBase databases, miRNA-targeted binding mrnas were predicted to be incorporated into ceRNA network construction only if miRNA-mRNA regulatory relationships were present in all three databases. The final 3 differential mirnas, through 66 miRNA-mRNA regulatory axes, predicted 65 mrnas. The 65 predicted mRNAs were intersected with 382 differential mRNAs from high throughput sequencing, resulting in 4 differential mRNAs (FOS, SLC6A4, PTK2 and KLF 4). The final formation of the ceRNA regulatory network of 13lncRNA-2miRNA-4mRNA was achieved using the Cytoscape 3.8.0 software (see Table 1 and FIG. 11). Based on the regulatory mechanism of the ceRNA network, and in combination with the prior literature, it was primarily determined that the inventive Chinese medicinal composition may exert therapeutic effects by modulating the lncRNA NEAT1/miR-139-5p/Fos axis (FIG. 12).
TABLE 1 ceRNA regulatory network of the inventive Chinese medicinal composition
1.9 to further verify whether the Chinese medicinal composition of the present invention delays the progression of atherosclerosis and renal insufficiency through lncRNA NEAT1/miR-139-5p/Fos axis, 20 patients from the treatment group were additionally selected for qRT-PCR verification.
RNA reverse transcription: reverse transcription of lncRNA was performed using lncRcute lncRNA First-Strand cDNA Kit. Reverse transcription of miRNA was performed using the miRcute Plus miRNA First-Strand cDNA Kit. Reverse transcription of mRNA was performed using the FastKing gDNA Dispelling RT SuperMix kit.
Primer design: primer Premier 5.0 software is used for designing Primer sequences of lncRNA NEAT1, fos, caspase 3, caspasee 9 and reference ACTB, and the required primers are synthesized by Beijing Saint gene technology Co. In addition, U6 and miR-139-5p primers were purchased from Beijing Tiangen Biochemical technology Co., ltd, and the primer sequences are shown in Table 2.
TABLE 2 primer sequences
Quantitative real-time reverse transcription polymerase chain reaction: the analysis was performed using an ABI7900HT fluorescent quantitative PCR instrument, lncRNA was reacted using a lnRcute lncRNA qPCR Kit (SYBR Green) kit, miRNA was reacted using a miRcute Plus miRNA qPCR Kit (SYBR Green) kit, and mRNA was reacted using a Power SYBR Green PCR Master Mix kit.
The results show (as shown in fig. 13), the expression of lncRNA net 1 (p=0.012) and Fos (p=0.033) can be significantly reduced and the expression of miR-139-5P (p=0.028) can be increased by the treatment of the traditional Chinese medicine composition. The qRT-PCR result is consistent with the result of transcriptome high-throughput sequencing, and the traditional Chinese medicine composition is preliminarily proved to have a therapeutic effect on patients with atherosclerosis and renal insufficiency possibly through intervention of the lncRNA NEAT1/miR-139-5p/Fos axis. Fos, an important component of activator protein transcription factor 1 (activator protein transcription factor-1, AP-1), can regulate apoptosis, and we further verify the expression of Caspase 3 and Caspase9 in patients. The study shows that the Chinese medicinal composition can obviously reduce the level of Caspase 3 (P=0.038), has the trend of inhibiting the expression of Caspase9, and has no statistical difference (P=0.095).
1.10 Elisa assay of plasma Fos protein: the expression level of plasma Fos before and after treatment was detected using the Human-c-Fos ELISA Kit.
Experimental results:
qRT-PCR has preliminarily confirmed that the lncRNA NEAT1/miR-139-5p/Fos axis may be an action target point for delaying atherosclerosis and renal insufficiency progression of the traditional Chinese medicine composition, and the traditional Chinese medicine composition can obviously reduce the mRNA level of Fos of a patient. We detected the expression of Fos protein in the plasma of patients before and after treatment with the Chinese medicinal composition of the present invention by ELISA technique (FIG. 14). The results show that, for 5 patients sequenced, the traditional Chinese medicine composition provided by the invention has a tendency of reducing the blood plasma Fos protein level, but has no statistical difference (P=0.064), the sample size is further enlarged, the analysis of 20 patients in a verification group shows that the traditional Chinese medicine composition provided by the invention can obviously reduce the blood plasma Fos protein level of the patients (P= 0.04995), and the combined analysis of the patients in the sequencing group and the verification group also shows that the traditional Chinese medicine composition provided by the invention can obviously reduce the blood plasma Fos protein level of the patients (P=0.008).
1.11 cell resuscitation and routine maintenance: the frozen cells were removed and placed in a 42℃water bath, quickly thawed within 1min, and placed in a 100mm petri dish for culturing. 24h after cell resuscitation, changing fresh growth medium, adding 0.25% pancreatin when cell density is increased to above 80%, digesting for 2min at room temperature, and adding growth medium to terminate reaction after cell is fully digested and suspended. Centrifuging in a centrifuge for 5min, and culturing the cell sediment with growth medium, or freezing the cell sediment after re-suspending with freezing solution for later use. 293 cell growth medium: 90% DMEM medium+10% fetal bovine serum; HUVEC cell growth medium: 90%1640 medium+10% foetal calf serum; HK-2 cell growth Medium: 90%1640 medium+10% foetal calf serum.
1.12 Transfection of 293 cells: after 293 cells were terminated by conventional digestion, the cell density was adjusted to 5X 104 cells/ml, and when the cell confluency reached 70% or more, solution A (100. Mu.l serum-free medium per well reaction system+Lipofectamine 2000 transfection reagent diluted 25-fold) and solution B (100. Mu.l serum-free medium per well reaction system+2. Mu.g plasmid) were prepared under aseptic conditions, and mixed at room temperature for 5min. Mixing the solution A and the solution B, and reacting for 15min at room temperature. Cell transfection groups are shown in table 3.
TABLE 3 transfection of 293 cells
1.13 double luciferase reporter detection: and detecting the Firefly luminescence value and the internal reference Renilla luminescence value by a luminescence instrument, and calculating the luminescence ratio and the relative expression quantity.
(1) Targeting binding of lncRNA NEAT1 to miR-139-5p
Studies have found that when lncRNA nea 1 wild type 293 cells were transfected with miR-139-5P mimic, the luminescence was significantly reduced (P < 0.001), whereas when lncRNA nea 1 mutant 293 cells were transfected with miR-139-5P mimic, no significant change in luminescence was caused (p=0.549), fig. 15. In addition, transfection of miR-139-5P mic into 293 cells at baseline levels did not cause significant changes in luminescence values (p=0.137). Thus, miR-139-5p can be targeted to bind with lncRNA NEAT1 and can inhibit the expression of lncRNA NEAT1, and possible binding sites of the two are shown in FIG. 16.
(2) Targeting binding of miR-139-5p to Fos
Studies have also found that when Fos wild-type 293 cells are transfected with miR-139-5P mimc, the luminescence value is significantly reduced (P < 0.001), whereas when Fos mutant 293 cells are transfected with miR-139-5P mimc, no significant change in luminescence value is caused (p=0.155). In addition, transfection of miR-139-5P mic into 293 cells at baseline levels did not cause significant changes in luminescence values (p=0.576), fig. 17. Thus, we demonstrate by dual luciferase reporter that miR-139-5p can bind to Fos targeting and is able to inhibit expression of Fos, the site of which is likely to bind is shown in figure 18.
1.14 non-toxic concentration screening of the Chinese medicinal composition: the absorbance of the HUVEC and HK-2 cells under the action of the Chinese medicinal composition of the invention with different concentrations (action concentrations: 400, 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78 and 0 mug/ml) is read by an enzyme-labeled instrument at 450nm for 24 hours by using the Chinese medicinal composition with different concentrations, and the cell inhibition rate is calculated. Cell inhibition ratio = [ (control OD value-experimental OD value)/control OD value ] ×100%. Analyzing the cell proliferation condition of each group of cells, and screening the maximum non-toxic concentration C1 of the traditional Chinese medicine composition to HUVEC cells and the maximum non-toxic concentration C2 to HK-2 cells.
Experimental results:
the present study applied 400. Mu.g/ml, 100. Mu.g/ml, 50. Mu.g/ml, 25. Mu.g/ml, 12.5. Mu.g/ml, 6.25. Mu.g/ml, 3.12. Mu.g/ml, 1.56. Mu.g/ml and 0.78. Mu.g/ml of the present invention to HUVEC and HK-2 cells, respectively, and the results of the CCK8 assay showed that the cell inhibition rate gradually decreased with decreasing concentration of the present invention, the maximum non-toxic concentration of the present invention to HUVEC cells was 3.12. Mu.g/ml, and the maximum non-toxic concentration of the present invention to HK-2 cells was 1.56. Mu.g/ml.
TABLE 4 non-toxic concentration screening results of the inventive Chinese medicinal compositions
1.15 Effect of the Chinese medicinal composition of the present invention on HUVEC and HK-2 cell proliferation
HUVEC and HK-2 cell model: cell grouping, group a: HUVEC cells, normal control group; group B: HUVEC cells, H 2 O 2 A model group; group C: HUVEC cells, H 2 O 2 +a therapeutic group of chinese medicinal compositions; group D: HK-2 cells, normal control; group E: HK-2 cells, H 2 O 2 A model group; group F: HK-2 cells, H 2 O 2 +chinese medicinal composition treatment group. HUVEC cells were pre-added with C1 (3.12 μg/ml) concentration of the Chinese medicinal composition for 23H, and 100 μM H was added to groups B and C 2 O 2 Acting for 1h; similarly, after the HK-2 cells were previously added with the C2 (1.56. Mu.g/ml) concentration of the Chinese medicinal composition and acted for 23 hours, 600. Mu.M H was added to groups E and F 2 O 2 The reaction time is 1h.
Cell proliferation assay: after the preparation of each cell model, 10 μl of CCK8 solution was added to each well, and the mixture was thoroughly mixed and reacted at 37deg.C for 2 hours. And (3) using an enzyme-labeled instrument to read an OD value at 450nm, calculating a cell inhibition rate, and analyzing the influence of the traditional Chinese medicine composition on the proliferation of each group of cells.
Research findings (see table 5 and fig. 19): h 2 O 2 The OD values were significantly reduced in the induced HUVEC cell model group compared to the control group (P<0.001 The average inhibition rate of cells reaches 58.89 percent, the proliferation level of cells is inhibited, and compared with a model group, the treatment group of the traditional Chinese medicine composition can obviously raise the OD value (P)<0.001 Improving cell survival and reducing H 2 O 2 Damage to HUVEC cells. In the case of HK-2 cells, H 2 O 2 Compared with the model group, the induced HK-2 cells can obviously reduce the OD value (P=0.002), the average inhibition rate of the cells reaches 37.02%, the proliferation level of the cells is inhibited, and compared with the model group, the treatment group of the traditional Chinese medicine composition can obviously raise the OD value (P=0.029), and the survival rate of the cells is improved. The Chinese medicinal composition can relieve H 2 O 2 Inhibition of HUVEC and HK-2 cell proliferation increases cell survival.
TABLE 5 Effect of the inventive Chinese medicinal composition on proliferation of HUVEC and HK-2 cells of each group
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Note that: in comparison with the control group, * P<0.05， ** P<0.01; in contrast to the set of models, # P<0.05， ## P<0.01。
1.16 flow cytometry detection: taking 100 mu l of diluted dye reaction liquid (ROS index dilution ratio is 1:100; apoptosis index dilution ratio is 1:20) for each index, re-suspending cells, and performing light-shielding reaction for 30min at normal temperature. 400. Mu.l of PBS was added to each well, and after resuspension of the cells, the cells were examined using a flow cytometer. 10000 ROS index collection cells per time, channel FL1; apoptosis index the total cell mass was 10000 per time, annwxinV-PE as channel FL2 and 7-AAD as channel FL4. Data were analyzed using CellQuest software.
(1) ROS level
Flow cytometry was used to detect ROS levels in each group. Research has found (see Table 6 and FIG. 20) that, for HUVEC cells, H 2 O 2 Induction significantly increased HUVEC cellular ROS levels (P<0.001 While the traditional Chinese medicine composition can obviously reduce H after intervention 2 O 2 ROS levels in induced HUVEC cells (p=0.001); for HK-2 cells, H 2 O 2 Induction significantly increased ROS levels in HK-2 cells (P<0.001 Compared with the model group, the treatment group of the traditional Chinese medicine composition can obviously reduce H 2 O 2 ROS levels in induced HK-2 cells (P<0.001). Therefore, the traditional Chinese medicine composition can inhibit H 2 O 2 Induced synthesis of ROS in HUVEC and HK-2 cells.
TABLE 6 Effect of the inventive Chinese medicinal composition on ROS levels in the HUVEC and HK-2 cells of the groups
| Group of | ROS fluorescence intensity |
| HUVEC control group | 82.79±0.86 |
| HUVEC model group | 532.04±83.40 ** |
| HUVEC treatment group | 270.44±9.79 ## |
| HK-2 control group | 170.36±0.24 |
| HK-2 model group | 497.71±5.95 ** |
| HK-2 treatment group | 405.23±1.71 ## |
Note that: in comparison with the control group, * P<0.05， ** P<0.01; in contrast to the set of models, # P<0.05， ## P<0.01。
(2) Apoptosis of cells
Flow cytometry was used to analyze the proportion of early apoptotic cells, late apoptotic cells, and total apoptotic cells in each group of cells. Research shows (as shown in fig. 21 and 22), H 2 O 2 The induced HUVEC cells can significantly increase the early apoptosis rate (P) of HUVEC cells compared with the control group<0.001 Rate of late apoptosis (P)<0.001 Total apoptosis rate (P)<0.001 After the treatment of the Chinese medicinal composition, the treatment group can obviously reduce the early apoptosis rate (P) of HUVEC cells compared with a model group<0.001 Rate of late apoptosis (P)<0.001 Total apoptosis rate (P)<0.001). For HK-2 cells, H 2 O 2 The induced HK-2 cells can significantly increase the early apoptosis rate (P) of the HK-2 cells compared with the control group<0.001 Rate of late apoptosis (P)<0.001 Total apoptosis rate (P)<0.001 After the treatment of the traditional Chinese medicine composition provided by the invention, the treatment group obviously reduces the late apoptosis rate (P=0.004) and the total apoptosis rate (P=0.001) compared with the model group, and has no obvious influence on the early apoptosis rate (P=0.065) of HK-2 cells. The Chinese medicinal composition has the effect of resisting H 2 O 2 Inducing apoptosis of HUVEC and HK-2 cells.
1.17Tunel staining: after completion of each group of model preparation, the supernatant was discarded and the cells were washed 2 times with PBS. After adding 500. Mu.l of the cell fixative, the cells were fixed at room temperature for 15min or overnight at 4℃and washed 2 times with PBS. Adding 1:200 diluted Tunel reagent and 1:200 dilution of DAPI reagent, at room temperature, light shielding reaction for 30 minutes, with PBS washing cells several times. Each group of cells was observed under a fluorescence microscope, each group was photographed 1 at 100-fold, photographed 6 at 200-fold, and apoptosis of each group of cells was detected and analyzed.
Experimental results:
and (3) analyzing the apoptosis condition of each group of cells by adopting a Tunel staining technology, wherein green fluorescence is Tunel-stained apoptotic cell nuclei, and blue fluorescence is DAPI-stained total cell nuclei. Research has found that H 2 O 2 Induced HUVEC cells resulted in a significant increase in green fluorescence spots and an increase in apoptosis index (P<0.001 After the treatment of the traditional Chinese medicine composition, compared with a model group, the green fluorescence point number of the treatment group is obviously reduced, and the apoptosis index is reduced (P)<0.001 The traditional Chinese medicine composition can obviously reduce H 2 O 2 Induce apoptosis index of HUVEC cells, and exert anti-HUVEC apoptosis effect, see FIG. 23 and FIG. 24.
For HK-2 cells, H 2 O 2 The induced HK-2 cells lead to a remarkable increase in green fluorescence point number, and the apoptosis index is increased (P=0.0017), so that the treatment group can reduce the green fluorescence quantity and the apoptosis index (P= 0.0241) compared with the model group after the treatment by the traditional Chinese medicine composition provided by the invention, and the anti-HK-2 cell apoptosis effect is exerted, as shown in fig. 25 and 26.
1.18qRT-PCR detection: in qRT-PCR detection, total RNA extraction, RNA reverse transcription, primer sequences, and qRT-PCR reaction conditions and operation steps are the same as those in 1.4 and 1.9.
Research shows (as shown in fig. 27 and 28), H 2 O 2 Compared with the control group, the induced HUVEC cell oxidative damage model can significantly increase lncRNA NEAT1 (P=0.040) and Fos (P<0.001 Level of miR-139-5P (P)<0.001 Compared with the model group, the Chinese medicinal composition treatment group can reduce the level of lncRNA NEAT1 (P=0.003) and Fos (P=0.012) and increase the level of miR-139-5P (P=0.026). In terms of expression of apoptosis-related genes Caspase 3 and Caspase9, H 2 O 2 ModelThe group can significantly increase Caspase 3 (p=0.040) and has a tendency to increase Caspase9 (p=0.081) compared to the control group, but there is no statistical significance. After the treatment by the traditional Chinese medicine composition provided by the invention, compared with a model group, the levels of Caspase 3 (P=0.008) and Caspase9 (P=0.030) can be obviously reduced in the treatment group. Likewise, H 2 O 2 Compared with a control group, the induced HK-2 cell oxidative damage model can also obviously raise the level of lncRNA NEAT1 (P=0.016) and Fos (P=0.004) and lower the level of miR-139-5P (P=0.014); compared with the model group, the Chinese medicinal composition treatment group can reduce the level of lncRNA NEAT1 (P=0.025) and Fos (P=0.011), and increase the level of miR-139-5P (P=0.036). In terms of apoptosis-related gene Caspase 3 and Caspase9 expression, the model group significantly increased Caspase 3 levels (p=0.007) and Caspase9 levels (p=0.019) compared to the control group. The treatment group significantly reduced Caspase 3 expression (p=0.046) and Caspase9 (p=0.026) compared to the model group.
1.19Western Blot detection: the method comprises the steps of protein extraction, protein quantification by a BCA method, western Blot protein detection and Western Blot detection of internal reference proteins, wherein an eBlot exposure instrument is used for exposure, the exposure time is set to be 10s, pictures with proper exposure time are selected, and the grey values of the proteins are analyzed by Image J software.
The study found that, compared with the control group, H 2 O 2 Induced HUVEC cells resulted in significant increases in Fos protein (p=0.029), AP-1 protein (p=0.008), caspase 3 protein (p=0.030) and Caspase9 protein (p=0.025); after the traditional Chinese medicine composition is dried and predicted, compared with a model group, the levels of Fos protein (P=0.037), AP-1 protein (P=0.040), caspase 3 protein (P=0.025) and Caspase9 protein (P=0.013) in a treatment group can be remarkably reduced, as shown in figure 29.
H against HK-2 cells, compared with the control group 2 O 2 Induced HK-2 cells significantly increased AP-1 protein (p=0.004), caspase 3 protein (p=0.022) and Caspase9 protein (P<0.001 Level) without significant differences for Fos protein (p=0.895); compared with the model group, the treatment group of the traditional Chinese medicine composition can obviously reduce the level (P=) of the AP-1 protein0.007 Caspase 3 protein level (P)<0.001 Caspase9 protein levels (p=0.001) and no significant differences for Fos protein (p=0.121), see figure 30.
The statistical method used in this embodiment: the SPSS Statistics 20.0 is adopted for data statistical analysis, and if the data accords with normal distribution, two independent sample t-tests or paired sample t-tests are adopted; if the normal distribution is not met, a Man-Whitney U nonparametric test or a Wilcoxon rank sum test is adopted. The comparison between groups is performed by using chi-square test, correction chi-square test, fisher accurate test, mcNemar test or single factor ANOVA. In qRT-PCR studies, the relative expression amount of genes was analyzed using 2 -△△CT The method. Delta Ct= (CT) Target gene -CT Reference gene ) Post-treatment- (CT) Target gene -CT Reference gene ) Before treatment. All tests used double sided test, P<0.05 indicates that the difference is statistically significant.
Knot (S)
The Chinese medicinal composition can obviously improve the clinical curative effects of patients with atherosclerosis and renal insufficiency, and the mechanism of the Chinese medicinal composition is to lighten H by regulating and controlling the lncRNA NEAT1/miR-139-5p/Fos axis 2 O 2 Damage to HUVEC and HK-2 cells, inhibit ROS formation, reduce expression of AP-1, caspase 3 and Caspase9, and inhibit apoptosis of HUVEC and HK-2 cells, thereby playing roles in delaying atherosclerosis progression and protecting kidney.
It should be understood that the foregoing examples of the present invention are provided merely for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention, and that various other changes and modifications may be made therein by one skilled in the art without departing from the spirit and scope of the present invention as defined by the appended claims.
Claims (9)
1. The traditional Chinese medicine composition for treating acute coronary syndrome combined renal insufficiency is characterized by being prepared from the following raw materials in parts by weight: 6-40 parts of cistanche, 10-40 parts of medlar, 3-20 parts of red paeony root, 6-20 parts of allium macrostemon, 3-15 parts of ginseng and 6-30 parts of ligusticum wallichii.
2. The traditional Chinese medicine composition according to claim 1, which is characterized by being prepared from the following raw materials in parts by weight: 30 parts of cistanche, 40 parts of medlar, 15 parts of red paeony root, 10 parts of allium macrostemon, 10 parts of ginseng and 10 parts of ligusticum wallichii.
3. The traditional Chinese medicine composition according to claim 1, which is characterized by being prepared from the following raw materials in parts by weight: 40 parts of cistanche, 30 parts of medlar, 20 parts of red paeony root, 20 parts of allium macrostemon, 15 parts of ginseng and 30 parts of ligusticum wallichii.
4. The traditional Chinese medicine composition according to claim 1, which is characterized by being prepared from the following raw materials in parts by weight: 6 parts of cistanche, 10 parts of medlar, 3 parts of red paeony root, 6 parts of allium macrostemon, 3 parts of ginseng and 6 parts of szechuan lovage rhizome.
5. A method for preparing a traditional Chinese medicine composition for treating acute coronary syndrome complicated with renal insufficiency according to any one of claims 1 to 4, characterized in that the preparation method comprises:
pulverizing the raw materials for preparing the traditional Chinese medicine composition, mixing, adding 6-10 times of water for soaking for 0.5-2 hours, decocting for 2-3 times, each time for 40-60 minutes, mixing decoctions, filtering, and concentrating the filtrate under reduced pressure until each milliliter of liquid medicine is equivalent to 0.2-3g of crude drug, thus obtaining the traditional Chinese medicine composition; or,
crushing Ginseng radix to obtain Ginseng radix, reflux-extracting with 6-10 times of 60-80% ethanol water solution for 2-3 times (each for 1-2 hr), filtering, collecting the combined filtrates, recovering ethanol, and concentrating the filtrate under reduced pressure to obtain Ginseng radix ethanol extract containing 0.1-1g crude drug per ml of medicinal liquid; mixing the residue with other materials, decocting with 6-10 times of water for 2-3 times for 40-60 min, mixing decoctions, filtering, mixing filtrate with the Ginseng radix ethanol extract, and concentrating into medicinal liquid of 0.2-3 g/ml; or,
pulverizing Ginseng radix into fine powder; mixing the other raw materials, decocting with 6-10 times of water for 2-3 times, each time for 40-60 min, mixing decoctions, filtering, concentrating the filtrate under reduced pressure until the concentration of each milliliter of liquid medicine is equivalent to 0.2-3g of crude drug, and adding Ginseng radix fine powder into the concentrated solution, and mixing.
6. A medicament for treating acute coronary syndrome complicated with renal insufficiency, which is characterized in that the medicament takes the traditional Chinese medicine composition as an active ingredient.
7. The medicament of claim 6, further comprising a pharmaceutically acceptable excipient.
8. The medicament according to claim 6, wherein the medicament is in the form of decoction, granule, pill, capsule, tablet, powder or oral liquid.
9. Use of a Chinese medicinal composition according to any one of claims 1-4 in the manufacture of a medicament for the treatment of acute coronary syndrome complicated with renal insufficiency.
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